Yasusuke Masuda

Carbon tetrachloride-induced lipid peroxidation of rat liver microsomes in vitro.

Abstract Characteristics of carbon tetrachloride-induced lipid peroxidation of rat liver microsomes and effect on microsomal enzymes were studies in vitro . Microsomes isolated from well-perfused livers and washed with EDTA-containing medium exhibited low endogenous lipid peroxidation when incubated in a phosphate buffer (> 0.1 M) in the presence of NADPH, whereas carbon tetrachloride stimulated to a great extent the peroxidation under these conditions. The stimulation was dependent on the concentration of NADPH, neither NADH nor ascorbic acid being replaced. The stimulatory action by bromotrichloromethane was more marked than that by carbon tetrachloride, however chloroform had no stimulatory action. N,N -Diphenyl- p -phenylene diamine, diethyldithiocarbamate and disulfiram inhibited carbon tetrachloride-induced lipid peroxidation in low concentrations. Inhibitions by thiol compounds and EDTA were weaker. Ferricyanide, cytochrome c and vitamine K 3 inhibited the stimulation by carbon tetrachloride while no inhibition was seen with carbon monoxide. An increase in the degree of carbon tetrachloride-induced lipid peroxidation resulted in a coincidental decrease in microsomal cytochrome P-450 content accompanying a parallel loss in aminopyrine demethylase activity, while NADH-ferricyanide dehydrogenase and NAD(P)H-eytochrome c reductase activities, and cytochrome b 5 content remained unaffected. Similar results were obtained when microsomes were peroxidized with NADPH in combination with ferric chloride and pyrophosphate. Regarding the mechanism of hepatotoxic action of carbon tetrachloride, these results support the hypothesis of lipid peroxidation.

Protective effect of diethyldithiocarbamate and carbon disulfide against liver injury induced by various hepatotoxic agents.

Diethyldithiocarbamate (DTC) and carbon disulfide (CS2), at nearly equimolar oral dose levels, protected mice against liver damage induced by carbon tetrachloride, chloroform, bromotrichloromethane, thioacetamide, bromobenzene, furosemide, acetaminophen, dimethylnitrosamine and trichloroethylene, as evidenced by the suppression of elevations in plasma GPT activity and liver calcium content, and of histopathological alterations. Both agents also prolonged hexobarbital sleeping time and zoxazolamine paralysis time in mice. DTC and SC, alone, given orally, decreased microsomal metabolism of several substrates (aniline, p-nitroanisole, hexobarbital, zoxazolamine, aminopyrine and 3,4-benzopyrene), CC14-induced lipid peroxidation, and cytochrome P-450 content. The loss of microsomal drug-metabolizing enzyme activity was also observed in the experiments in vitro using liver slices and isolated microsomes. Since a characteristic common to such diverse hepatotoxins is that they require metabolic activation before exhibiting hepatotoxicity, the protective mechanisms of DTC and CS2 may involve their interference with the process of metabolic activation of these hepatotoxins. The protective action of DTC may be mediated almost entirely through CS2 when administered orally and at least partly with parenteral administration, since, in CCl4-induced liver injury, DTC was most effective when given orally, while the action of CS2 was less dependent on the route of administration. Thus CS2 and CS2-producing agents in vivo such as dithiocarbamate derivatives and disulfiram may modify toxicological and pharmacological effects of foreign compounds by inhibiting microsomal drug-metabolizing enzyme activity in the liver.

Protective action of diethyldithiocarbamate and carbon disulfide against acute toxicities induced by 1,1-dichloroethylene in mice.

In male mice of ddY strain, a single dose of 1,1-dichloroethylene (1,1-DCE, 0.1 ml/kg, ip) produced severe renal damage at 24 hr, as evidenced by elevations in plasma urea nitrogen concentration and kidney calcium content and by massive renal tubular necrosis, while hepatic damage was less severe. A precipitous decrease in body temperature started as early as 30 min after administration of 1,1-DCE and lasted for 24 hr. Glutathione concentrations decreased in the liver and kidney, with a rebound increase seen in the former but not in the latter tissue. In carbon tetrachloride-poisoned mice, the renal toxicity of 1,1-DCE was markedly potentiated. Pretreatment with either diethyldithiocarbamate (DTC) or carbon disulfide (CS2) blocked all of these 1,1-DCE-induced toxic manifestations in normal and carbon tetrachloride-poisoned mice. Both agents, however, did not prevent the hypothermia induced by monochloroacetic acid or chloroacetyl chloride, proposed active metabolites of 1,1-DCE. Since DTC and CS2 inhibited hepatic and renal microsomal drug metabolizing enzyme activities (Masuda and Nakayama, 1982, 1983), it is probable that the protective action of DTC and CS2 against renal and hepatic injury induced by 1,1-DCE may be due to an inhibition of the metabolic activation of 1,1-DCE to its proposed epoxide in each organ. The action of DTC given po may be mediated by CS2 produced in the stomach. The hypothermia induced by 1,1-DCE may not result from a direct action of 1,1-DCE per se, but by its metabolites.

Prevention of butylated hydroxytoluene induced lung damage by diethyldithiocarbamate and carbon disulfide in mice

Diethyldithiocarbamate (DTC) and carbon disulfide (CS2), at nearly equimolar doses (po), prevented mice from lung injury induced by butylated hydroxytoluene (BHT), as evidenced by suppression of increased lung weight and total DNA content as well as by histopathologic observations. CS2 pretreatment dose dependently decreased the amount of covalently bound [ring-14C]BHT to lung macromolecules in vivo. A slight, but significant, loss of lung GSH observed early after BHT administration was also prevented. The lung microsomal fraction exhibited NADPH-dependent covalent binding of BHT in vitro; this was inhibited completely by carbon monoxide and slightly by SKF-525A. This NADPH-dependent binding was suppressed in lung microsomes isolated from CS2-treated mice. CS2 also reduced various drug metabolizing enzyme activities and the cytochrome P-450 content of the lung microsomal fraction. These results support the metabolic activation hypothesis for BHT-induced lung damage, and the preventive action of CS2 and DTC may be due to an inhibition of this bioactivation step. Possible sites of the metabolic activation of BHT and its inhibition by CS2 are discussed.

Carbon tetrachloride-induced cell death in perfused livers from phenobarbital-pretreated rats under hypoxic conditions and various ionic milieu: Further evidence for calcium-dependent irreversible changes

The role of Ca2+ in the initiation of carbon tetrachloride (CCl4) hepatotoxicity was studied using perfused livers isolated from phenobarbital-pretreated rats in a single-pass system. Krebs-Henseleit bicarbonate buffer containing 1.3 mM CaCl2 (KHB) was the regular ionic milieu. In the liver perfused with fructose-supplemented regular KHB equilibrated with 95% N2-5% CO2, infusion of 0.5 mM CCl4 caused an early uptake of Ca2+ coupled with K+ leakage and Na+ uptake within the infusion time of 30 min, which was followed by a marked lactic dehydrogenase (LDH) leakage into the effluent perfusate and further Ca2+ uptake by the liver. With Ca(2+)-free medium, the prenecrotic K+ leakage and the successive LDH leakage were suppressed markedly. However, a perfusate exchange from regular to Ca(2+)-free KHB at the end of the prenecrotic stage did not protect against the LDH leakage, and the perfusate exchange conversely did not produce LDH leakage. Perfusion of the liver with high K+(Cl-) medium under 20% O2 markedly suppressed CCl4-induced LDH leakage even in the presence of Ca2+, whereas once CCl4 had acted under regular KHB perfusion, changing the medium to high K+ did not further prevent the LDH leakage. High K(+)-lactobionic acid medium containing Ca2+ and supplemented with fructose also suppressed LDH leakage under 95% N2 without the accompanying prenecrotic Ca2+ uptake. However, a change of the medium after CCl4 infusion to regular KHB containing Ca2+ caused LDH leakage and K+ leakage, with Ca2+ uptake. The prevention of LDH leakage in a different ionic milieu may not be due to suppression of CCl4 bioactivation, since the liver cytochrome P450 content decreased to a similar extent. These findings suggest that entry of extracellular Ca2+ into hepatocytes coupled with K+ leakage and Na+ entry is a prerequisite for CCl4-induced hepatocyte death and that association of Ca2+ with a CCl4-derived radical-mediated process may be necessary for early and irreversible plasma membrane damage.

Early, selective and reversible suppression of cytochrome P-450-dependent monooxygenase of liver microsomes following the administration of low doses of carbon disulfide in mice

The effects of carbon disulfide (CS2) on the liver microsomal drug-metabolizing enzyme system and other enzyme activities were studied 1 hr after the oral administration of 3-300 mg/kg of CS2 in mice. Considerable decreases in drug-metabolizing enzyme activities (such as hydroxylation of aniline, O-dealkylation of p-nitroanisole, 7-ethoxycoumarin and 7-ethoxyresorufin, and N-demethylation of N,N-dimethylaniline), NADPH-cytochrome P-450 reductase (but not NADPH-cytochrome c reductase), and P-450-associated peroxidase activities were already observed at 3 and 30 mg/kg of CS2, dose dependently. At the same dosage levels, the magnitudes of microsomal spectral changes induced by aniline and nicotinamide (type 2 substrates), but not those induced by hexobarbital and SKF-525A (type 1 substrates), were also reduced to a considerable extent. The degrees of these alterations were all greater than that of the measurable loss of P-450 content, i.e. the loss of functional activity of P-450 was much greater than simply expected from the apparent decrease in the hemoprotein content. Cytochrome b5 content and NADH-ferricyanide reductase activity were unchanged at 30 and 300 mg/kg of CS2, although NADH-cytochrome c reductase activity was increased at the latter dose. The following enzyme activities did not change significantly at up to 300 mg/kg of CS2: flavin-containing monooxygenase, UDP-glucuronyl transferase, glucose-6-phosphatase and heme oxygenase in microsomes, and glutathione S-transferases in the soluble fraction. Microsomal conjugated diene levels and liver glutathione content were also unchanged. These observations support the theory that P-450 is a sensitive and selective site for CS2 action, where CS2 itself is bioactivated. It was also shown that the loss of P-450 was reversible after a single, or repeated, administration of CS2.

Protective action of diethyldithiocarbamate and carbon disulfide against renal injury induced by chloroform in mice

Oral administration of diethyldithiocarbamate (DTC) and carbon disulfide (CS2) protected mice against CHCl3-induced kidney injury, as evidenced by normalization of delayed plasma phenolsulfonphthalein clearance, suppression of increased kidney calcium content and prevention of renal tubular necrosis. In CCl4-treated mice, in which liver microsomal monooxygenase activities were decreased markedly, and kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities were increased to about twice those of the untreated mice, renal toxicity of CHCl3 was greatly potentiated, and the latter effect was also blocked by both agents. DTC and CS2 per se markedly decreased kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities at 1 hr after oral administration, accompanying a moderate loss of cytochrome P-450 content, in both normal and CCl4-treated mice. The protection was not due to hypothermia, because pretreatment with DTC or CS2 (p.o.) also prevented the hypothermia induced by CHCl3. The mechanism of the protection may have involved inhibition of metabolic activation of CHCl3 in the kidney rather than in the liver.

Biphasic effects of oxethazaine, a topical anesthetic, on the intracellular Ca2+ concentration of PC12 cells

There have been few reports on the mechanism(s) of action of oxethazaine (OXZ) despite its potent local anesthetic action. Generally, local anesthetics (LAs) not only inhibit Na(+) channels but also affect various membrane functions. In the present study, using PC12 cells as a nerve cell model, the effects of OXZ on intracellular Ca(2+) concentration ([Ca(2+)](i)) were examined in relation to cytotoxicity and dopamine release. [Ca(2+)](i) was determined by the quin2 method. In resting cells, (6-10)x10(-5)M OXZ produced lactate dehydrogenase leakage, which was Ca(2+)-dependent, inhibited by metal Ca(2+) channel blockers, and preceded by a marked increase in [Ca(2+)](i). Some other LAs showed no cytotoxicity at these concentrations. In K(+)-depolarized cells, however, lower concentrations of OXZ (10(-6)-10(-7)M), that had no effect on resting [Ca(2+)](i), inhibited both the dopamine release and the increase of [Ca(2+)](i) in parallel. The inhibitory potency against the [Ca(2+)](i) increase was in the order of nifedipine>OXZ approximately verapamil>diltiazem, and OXZ acted additively on the Ca(2+) channel blockers. OXZ showed the least effect on K(+)-depolarization as determined by bisoxonol uptake. OXZ also inhibited the increase in [Ca(2+)](i) induced by S(-)-BAY K 8644, a Ca(2+) channel agonist. These observations suggested that low concentrations of OXZ interact with L-type Ca(2+) channels. The biphasic effects of OXZ on Ca(2+) movement may be due to a unique chemical structure, and may participate in and complicate the understanding of the potent pharmacological and toxicological actions of OXZ.

K+-driven sinusoidal efflux of glutathione disulfide under oxidative stress in the perfused rat liver

Tert‐butyl hydroperoxide (BHP), hydrogen peroxide and diamide caused a rapid and simultaneous release of glutathione disulfide (GSSG) and K+ in the isolated perfused rat liver. Both BHP‐induced effluxes were suppressed by prior depletion of hepatic glutathione, but not by co‐infusion of desferrioxamine which prevented lipid peroxidation and cell death. High K+ media decreased the GSSG efflux even though hepatic GSSG levels remained high. The GSSG and K+ effluxes were repeatable if cellular K+ recovered after a short BHP exposure. Ouabain inhibited the K+ re‐uptake and decreased the response to repeated BHP challenge. Thus, sinusoidal efflux of GSSG under oxidative stress may be driven by a K+ gradient.

Opioid peptide participates in post-tetanic twitch inhibition in guinea pig isolated ileum

The effects of a mixture of peptidase inhibitors, consisting of 100 nM each of amastatin, phosphoramidon, and captopril, on the twitch inhibitory response (0.1 Hz, 0.5 ms duration, maximum intensity) exerted by opioid peptides were investigated. The opioid peptides, Met-enkephalin (50-200 nM), dynorphin(1-13) (0.2-1 nM), and beta h-endorphin (20-100 nM) concentration-dependently inhibited the electrically evoked twitch response. In the presence of the mixture of peptidase inhibitors, the twitch inhibition evoked by Met-enkephalin was significantly increased; however, the twitch inhibition evoked by beta h-endorphin and dynorphin(1-13) was only slightly increased. These increases were abolished by naloxone (NLX; 100 nM). Inhibition of the twitch response (0.1 Hz, 0.5 ms duration, maximum intensity) induced after high frequency stimulation (10 Hz, 0.5 ms pulse width, maximum voltage for various lengths of time) (post-tetanic twitch inhibition) was investigated in isolated guinea pig ileal longitudinal muscles. The mixture of peptidase inhibitors, which did not affect the twitch response or ACh-contraction, increased post-tetanic twitch inhibition. This increase was abolished by naloxone (100 nM). These results suggested that the potentiated post-tetanic twitch inhibition evoked by the peptidase inhibitors in the ileum was due primarily to mu-ligand(s) rather than to the kappa-type of endogenous opioid ligand(s) released from opioidergic neurons.